This invention is concerned with a multivalent pneumococcal vaccine consisting of purified pneumococcal capsular polysaccharide with the "C" polysaccharide substantially absent. This invention is also concerned with the specific purification of each of 16 pneumococcal types which by Danish designation are types 1, 2, 3, 4, 6A, 6B, 7F, 8, 9N, 12F, 14, 18C, 19F, 20, 23F and 25, to yield the purified immunogenic polysaccharides of the invention.
Pneumococcal cultures of each type useful in this invention are stored and available worldwide from a great number of culture libraries. The American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, U.S.A. 20852, lists all the pneumococcal types of this invention as being freely available.
The 1978 ATCC catalogue designates these types as follows: (See Table I)
TABLE I ______________________________________ Danish Type U.S. Catalogue Nomenclature Nomenclature Number ______________________________________ 1 1 6301 2 2 6302 3 3 6303 4 4 6304 6A 6 6306 6B 26 6326 7F 51 10351 8 8 6308 9N 9 6309 12F 12 6312 14 14 6314 18C 56 10356 19F 19 6319 20 20 6320 23F 23 6323 25 25 6325 ______________________________________
The critical step in the preparation of a vaccine is purification of the immunogenic material such that extraneous material is removed without loss of those properties of the retained material that will cause the appropriate antibody production. Such properties of polysaccharide appear to reside in the retention of what may be termed the "native state configuration" of the polysaccharide.
Among those materials to be separated from the polysaccharide are proteins, nucleic acids and "C" polysaccharide. "C" polysaccharide is found in high concentration in Danish designation pneumococcal types 4, 7F, and 14.
"C" polysaccharide is a choline containing teichoic acid on the cell wall of pneumococcus and is species specific. It is further described in Tomasz, A., Science 157 694 (1967) and Brundish, D. E., and Baddeley, J., Biochem. J. 110 573 (1968) and Nosser, J. L., Tomasz, A., J. Biol. Chem. 245 #2, 287, (1970).
Nucleic acids (which absorb light at 260 MMU) are difficult to reduce to a satisfactory level in preparations of pneumococcal polysaccharides. This problem is in contradistinction to the situation presented by meningiococcal polysaccharide which is more easily purified while retaining immunogenicity. Meningiococcal polysaccharides may be purified by relatively harsh methods as shown in U.S. Pat. No. 3,636,192 to Gotschlich. There are 85 specific types of pneumococcus. These types are designated by both American and Danish numbering systems. Type designations cited herein are to the Danish numbering. Each type appears to require a particular method for eliminating contaminants but no single method is applicable to all types of pneumococcal polysaccharide. Further the specific proper method appears to be unpredictable. As exemplary of the different procedures used to purify various pneumococcal polysaccharides, some require a large volume of ethanol for precipitation, such as Type 7F which can be partially separated from nucleic acids by fractional precipitation as the nucleic acids are precipitated in the 30-50%* alcohol ranges using 3A alcohol. FNT [* % alcohol ranges refer to the volume of alcohol used related to the solutions original volume. 3A alcohol is 5% absolute methanol and 95% absolute ethanol. Absolute ethanol would behave in an essentially identical manner and is considered fully equivalent. Throughout this specification the term "alcohol" will designate 3A alcohol unless otherwise specified.]
With other types, such as Type 3, polysaccharides are precipitated in the 30-50% range thus alcohol is not effective as a separatory precipitant. In contrast, types 1, 8 and 12 can be separated from nucleic acids by carefully controlled amounts of protamine sulfate. With these types at an optimal concentration of protamine sulfate (0.02-0.20%), nucleic acids are precipitated and can be pelleted by high speed centrifugation. However, any excess protamine sulfate in the system beyond the minimum amount required to precipitate the constituent nucleic acid will additionally precipitate the polysaccharide. An example of another type of purification of pneumococcal polysaccharide is presented by the purification often used for Type 3 pneumococcus, which is difficult to separate from nucleic acid. If calcium acetate is substituted for sodium acetate as the electrolyte in a solution of Type 3 pneumococcal polysaccharide, the polysaccharide can be precipitated with a minimal amount of alcohol (10-12%). However, this method sometimes allows substantial amounts of nucleic acid to remain soluble in the supernatant phase. The behavior of various pneumococcal polysaccharide types in a reaction of the polysaccharide-nucleic acid mixtures with ammonium sulfate is also variable. Some polysaccharides are precipitated by ammonium sulfate salt at 50-60% saturation whereas others are not. Type 1 polysaccharide is not precipitated with ammonium sulfate whereas Type 3 and Type 4 may be separated to some degree from nucleic acids by 50% saturation with ammonium sulate. From the foregoing exposition and from the following references (Guy, R. C. W., How, J., Stacey, M., Heidelberger, M., J. Biol Chem. 242 21 (1967); Brown, R., J. Immunol. 37 455 (1939); Glaudemans, C. P. J., Treffers, H. P., Carbohydrate Res. 4, (1967); Kabat, E. A., Exp. Immunochemistry, Charles C. Thomas, publisher, pp. 838-842 (1967)) it can be seen that there is no one satisfactory method for the removal of contaminants from pneumococcal polysaccharide applicable to all types in view of the fact that there are 85 or more types of pneumococcus and the production of a practical vaccine usually requires a multivalent vaccine comprising polysaccharide fractions from many species of pneumococcus, each retaining a relatively native state configuration.
Another contaminant of pneumococcal polysaccharide is protein. Although alcohol precipitation is effective in reducing the level of protein contamination it is unable to reduce the contamination to a level satisfactory for a parenteral product. One method commonly employed to reduce the level of protein is to subject a mixture of pneumococcal polysaccharides and protein to organic solvents. For example, the "Sevag" procedure [Sevag, M. G. Biochem. Z., 272 419 (1934)] involves extraction of chloroform and butanol mixtures shaken vigorously for 4-6 hours and then subjected to low speed centrifugation. Denatured protein which collects at the interface can then be separated from the aqueous phase with the polysaccharides. However, this procedure is unsatisfactory as the extraction often adversely affects the pneumococcal polysaccharides causing their breakdown, depolymerization or loss of native state configuration. The result is polysaccharide that is not effective as an immunogen. Other procedure may be employed to reduce protein contamination such as ammonium sulfate precipitation and molecular sieving but such procedures are specific to each group of proteins and peptides among the many different sizes and types of proteins in the solution. Here again the variability of the polysaccharides, depending the on strain, determines the effectiveness the particular protein separatory step employed. Further, one many conclude that no one procedure is effective in purifying all pneumococcal capsular polysaccharides, and prediction of the behavior of a particular pneumococcal capsular polysaccharide appears impossible.
However, a number of methods of purifying pneumococcal capsular polysaccharide, with high purity and retention of immunogenic properties have now been discovered. These purifications have been specifically directed to the purification of 16 types of pneumococcus. These types are 1, 2, 3, 4, 6A, 6B, 7F, 8, 9N, 12F, 14, 18, 19F, 20, 23F, and 25 (Danish designation).